Summary Over the millennia, humans and their microbiota have coevolved a mutually beneficial relationship. Many essential aspects of host physiology, including immune system development and intestinal homeostasis, depend on interactions between cells of the intestine, the intestinal immune system, and the microbiota. For the intestinal immune system, these interactions require that it strike a balance between providing robust anti- microbial responses to pathogenic microbes while limiting inflammatory responses against the microbiota. To understand how this balance is achieved and maintained, it is critical to understand the intestinal microbes, immune cell populations, and signaling pathways involved. Data from our work and others suggest that CX3CR1 expressing mononuclear phagocytes (MNPs) detect signals from the microbiota and coordinate intestinal immune responses to promote epithelial integrity and reduce intestinal inflammation. This population of MNPs is highly phagocytic and is known to secrete both pro- and anti-inflammatory cytokines. In addition, this cell population has been found to be expanded in the colon of inflammatory bowel disease (IBD) patients as well as in mouse models of colitis. We hypothesize that CX3CR1+ MNPs recognize specific members of the microbiota, which in turn dictates their ability to mediate either protective or pathological responses. Changes in the commensal microbes encountered by CX3CR1+ MNPs would therefore be predicted to impact the function of these MNPs. As a hallmark of inflammatory conditions such as IBD is dysbiosis, or a shift in the composition of the microbiota, changes in the commensal microbes encountered by CX3CR1+ MNPs would directly impact their function. We have identified E. coli that are normal members of the mouse microbiota and offer different degrees of protection in mouse models of colitis. We will utilize this mouse commensal as well as E. coli human clinical isolates to understand how colonization with these microbes impacts the effector function of CX3CR1+ MNPs. We will further determine how these isolates support immune protection in models of colitis. Finally, with the goal of identifying microbial genetic elements which induce immune protection, we will utilize comparative genomics-based analysis to identify and subsequently target microbial gene clusters in these E. coli isolates that correlate with protective immunity. Our overall objective is to determine how specific members of the microbiota impacts intestinal immunity as the first step in understanding the regulation of intestinal homeostasis by the microbiota.